The basic antibiotics of the .beta.-lactam type are principally obtained by fermentation. Fungi of the genus Penicillium and Cephalosporium (Acremonium) are used for the production of raw material for .beta.-lactam antibiotics as Penicillin G, Penicillin V and Cephalosporin C. These fermentation products, also referred to as PenG, PenV and CefC, respectively, are the starting materials for nearly all currently marketed penicillins and cephalosporins. The side-chains of these compounds, phenylacetyl, phenoxyacetyl and aminoadipyl, respectively, are removed by cleavage of an amide linkage (deacylation), resulting in 6-aminopenicillanic acid (6-APA) in case of the two penicillin molecules and 7-aminocephalosporanic acid (7-ACA) in case of the cephalosporin. The particular enzymes which accomplish these conversions are referred to herein as "acylases" or "amidases". These denominations as used in this specification have the same meaning.
Also, the conversion of Cephalosporin G to 7-amino 3-deacetoxycephalosporanic acid (7-ADCA) is mentioned. However, Cephalosporin G (CefG) is not a fermentation product but is usually produced chemically from Penicillin G. The basic structures of the various penicillins and cephalosporins discussed above are shown in FIG. 1.
Synthetic manipulation to produce the various penicillins and cephalosporins of choice basically starts from 6-APA, 7-ACA and 7-ADCA, respectively.
The conversion of Penicillin G and Penicillin V to 6-APA may be performed both chemically and enzymatically. The classical way is the chemical cleavage, but enzymatic processes are preferred nowadays (for review, see Lowe [1]). Costs and environmental considerations are arguments in favour of an enzymatic process.
The cleavage of the CefC side-chain to 7-ACA is usually carried out chemically, according to the so-called imino-halide process. However, this process has serious disadvantages, since it is complex, requiring inter alia multiple steps, extremely low temperatures and expensive reagents.
The conversion of .beta.-lactam intermediates to the desired semi-synthetic antibiotics may also be performed chemically and enzymatically, the enzymatic route being basically preferred if a suitable enzyme is available. Penicillin acylases are such enzymes in a number of cases. The enzymatic conversion takes advantage of the fact that any enzymatic reaction is reversible, if the correct conditions are applied (Abbott B. J. [2]).
Various types of microorganisms have been proposed in the literature as acylase producing strains useful for the deacylation of .beta.-lactam derivatives obtained by fermentation and/or the acylation of 6-APA and 7-ACA to semi-synthetic .beta.-lactam antibiotics of choice. Examples of such acylase producing microorganisms are certain strains of the species Escherichia coli, Kluyvera citrophila, Proteus rettgeri, Pseudomonas sp., Alcaligenes faecalis, Bacillus megaterium, Bacillus sphaericus, and Arthrobacter viscosus.
According to the literature several types of acylases may be envisaged, based on their molecular structure and substrate specificity (Vandamme E. J. [3]).
Type-I acylases are specific for Penicillin V. These enzymes are composed of four identical subunits, each having a molecular weight of 35 kDa. A complete nucleotide sequence of the cloned gene from Bacillus sphaericus has been reported (Ollson A. [4]).
Type-II acylases all share a common molecular structure: these enzymes are heterodimers composed of a small subunit (.alpha.; 20-25 kDa) and a large subunit (.beta.; 60-65 kDa). With respect to the substrate specificity, Type-II acylases may be further divided into two groups:
Type-IIA acylases are very specific for Penicillin G. In general, they are not so much specific for the moiety adjacent to the nitrogen atom of the amide group (this might be a cephem group, a penem group, an amino acid, etc.), but the substrate specificity resides in the acyl moiety of the substrate. This acyl moiety must be very hydrophobic and is preferably benzyl or (short) alkyl. Examples of substrates which are not hydrolyzed by Type-IIA acylases are those with dicarboxylic acids as acyl moiety: succinyl, glutaryl, adipyl and also aminoadipyl, the side-chain of CefC. Examples of Type-IIA acylases are the enzymes from Escherichia coli, Kluyvera citrophila, Proteus rettgeri and Alcaligenes faecalis. Type-IIB acylases have been reported to be capable of hydrolyzing cephalosporins (including the desacetoxy-derivative) with succinyl, glutaryl and adipyl as an acyl moiety and even in one case CefC to a very limited degree (Shibuya Y. [5]; Matsuda A. [6]). So far these acylases have only been found in Pseudomonas species, and in certain strains of Bacillus megaterium and Arthrobacter viscosus.
The literature relates mainly to penicillin acylases. The synthetic potential of penicillin acylases, however, is limited due to the specificity of the enzyme. In more recent years also publications relating to Cephalosporin C acylases have appeared, but the activity of the reported enzymes was relatively low. No commercial enzymatic process for the conversion of Cephalosporin C to 7-ACA is available up to now, despite intensive efforts to find a suitable enzyme (cf. Walton R. B. [7]).
There is, therefore, a substantial interest in developing acylase enzymes which are highly efficient in deacylation/acylation reactions to produce desired chemical entities. Of particular interest are the enzymatic deacylation of .beta.-lactams and especially PenG, PenV and CefC, and derivatives thereof, to 6-APA and 7-ACA and derivatives, respectively, and the acylation of the latter compounds to produce semi-synthetic pencillins and cephalosporins of interest. It is of major importance in this connection to dispose of an efficient acylase enzyme which is capable of catalyzing the conversion of CefC (and derivatives) to 7-ACA (and derivatives).
The invention aims to provide such efficient enzymes.